What characteristics make up the most efficient PCR primers?

PCR primer design depends heavily on several factors, including Tm, length, GC content, secondary structure, and specificity. A wide range of primer Tms can be used in different PCR applications. While the functional Tm of the primer sequences will vary, it is important that all primer sequences within a single reaction have similar Tm values so that they will anneal properly. Ideally you want the Tm values for your primers to be +/-2°C of one another.

With regards to length, primers typically need to be at least 18 bases long in order to uniquely bind to their target among a heterogeneous mix of sequences. The length of the primer sequence will mostly be determined by the desired Tm.

GC content is also important as it will affect Tm and the secondary structures possible within a given primer sequence. GC percentage should ideally be between 35 and 65%. Strong secondary structures can prevent a primer from binding to its intended target. Both hairpins and dimers should be considered when analyzing secondary structures. Dimer strengths should have a Delta G value more positive than –9 kcal/mole and software predicted hairpin Tms should be 5°C below your annealing temperature.

Finally, it is recommended to BLAST your primer sequences to ensure target specificity.